Treatment of carcinomas using squalamine in combination with other anti-cancer agents

ABSTRACT

A method for treating a tumor includes a first treatment procedure using a conventional cancer treatment technique, and a second treatment procedure which includes administering an effective amount of squalamine. The first treatment procedure may be a treatment with one or more conventional cytotoxic chemical compounds. As examples, the cytotoxic chemical compound may be a nitrosourea (such as BCNU), cyclophosphamide, adriamycin, 5-fluorouracil, paclitaxel and its derivatives, cisplatin or other platinum containing cancer treating agents. The cytotoxic chemical compound and the squalamine may be administered by any suitable route. The first treatment procedure may take place prior to the second treatment procedure, after the second treatment procedure, or the two treatment procedures may take place simultaneously. In one example, the first treatment procedure (e.g., a one time intravenous dosage of BCNU) is completed before the second treatment procedure with squalamine begins. As an alternative, the first treatment procedure may be a conventional radiation treatment regimen.

This application claims priority to U.S. Provisional Application No.60/016,387, filed Apr. 26, 1996.

BACKGROUND OF THE INVENTION I. Information Relating to PreviousSqualamine Applications

This invention relates to various methods for using squalamine.Squalamine, having the structure illustrated in FIG. 1, is anaminosterol which has been isolated from the liver of the dogfish shark,Squalus acanthias. This aminosterol is the subject of U.S. Pat. No.5,192,756 to Zasloff, et al., which patent is entirely incorporatedherein by reference. Methods for synthesizing squalamine have beendevised, such as the methods described in WO 94/19366 (published Sep. 1,1994). This PCT publication is entirely incorporated herein byreference. This PCT application also relates to U.S. patent applicationSer. No. 08/023,347 (filed Feb. 26, 1993), which application also isentirely incorporated herein by reference. Additional methods forsynthesizing squalamine also are described in U.S. Provisional PatentAppln. No. 60/032,378 filed Dec. 6, 1996, which application also isentirely incorporated herein by reference.

U.S. patent application Nos. 08/416,883 (filed Apr. 20, 1995) and08/478,763 (filed Jun. 7, 1995) describe the use of squalamine as anantiangiogenic agent. These U.S. patent applications are entirelyincorporated herein by reference. Additional uses of squalamine (e.g.,as a sodium/proton exchanger (isoform 3), or NHE3, inhibiting agent andas an agent for inhibiting the growth of endothelial cells) andsqualamine synthesis techniques are disclosed in U.S. patent applicationNo. 08/474,799 (filed Jun. 7, 1995). This U.S. patent application alsois entirely incorporated herein by reference.

II. Information Relating to This Invention

About 50,000 new cases of CNS (central nervous system) tumors arediagnosed each year. Of these, about 35,000 are metastatic tumors (e.g.,lung, breast, melanomas) and about 15,000 are primary tumors (mostlyastrocytomas). Astrocytomas, along with other malignant gliomas (i.e.,cancers of the brain), are the third leading cause of death from cancerin persons between the ages of 15 and 34.

Treatment options for a patient with a CNS tumor are very limited.Currently, surgery is the treatment of choice. Surgery provides adefinite diagnosis, relieves the mass bulkiness of the tumor, andextends survival of the patient. The only post-surgery adjuvanttreatment which is known to work on CNS tumors is radiation, and it canprolong survival. Radiation treatment, however, has many undesirableside effects. It can damage the normal tissue of the patient, includingthe brain tissue. Radiation also can cause the patient to be sick (e.g.,nausea) and/or to temporarily lose their hair.

The other common post-surgery adjuvant cancer treatment, chemotherapy,is relatively ineffective against CNS tumors. Specifically, chemotherapyagainst CNS tumors with nitrosoureas is not curative. Many other cancertreating agents have been studied and tested, but generally they have aminimal effect on extending survival.

In view of these limited treatment options, the current prognosis forpersons with CNS tumors is not good. The median survival term forpatients with malignant astrocytomas having surgery and no adjuvanttreatment is about 14 weeks. Radiation therapy after surgery extends themedian to about 36 weeks. The current two year survival rate for allforms of treatment is less than 10%.

To maximize survival, it is critical to begin treatment in the earlystages of CNS tumor development. Typically, the extent of tumorangiogenesis (i.e., blood vessel formation) correlates with survival inthe patient. CNS tumors are among the most angiogenic of all humantumors. When the tumor is small, however, it is in an "avascular" phase,and its growth is restricted by a diffusion mechanism (i.e., the cellsreceive their nutrition, etc. by diffusion into the cell). In thisphase, the tumor is viable, but not growing, and it is unable to spread.Over time, however, angiogenesis begins and the tumor converts to a"vascular" phase. In this phase, perfusion replaces diffusion as thegrowth mechanism, and tumor growth is exponential (i.e., the tumor hasits own blood vessels to provide nutrients, etc.). Mitotic cells clusteraround new blood vessels and metastases occur in the vascular phase(i.e., the tumor can spread to other areas in the body). Therefore, bytreating the tumor early (before it reaches the vascular phase), one canhope to inhibit metastatic spread as well as control the primary tumor.

Other types of cancer also are difficult to combat by known cancertreatments. Lung cancer kills more Americans annually than the next fourmost frequently diagnosed neoplasms combined. Estimates for 1994indicate more than 170,000 new cases of lung cancer and approximately150,000 deaths (Boring et al.; CA Cancer J. Clin. 1994, 44: 7-26).Approximately 80% of primary lung tumors are of the non-small cellvariety, which includes squamous cell and large cell carcinomas, as wellas adenocarcinomas.

Single-modality therapy is considered appropriate for most cases ofearly and late stage non-small cell lung cancer (NSCLC). Early stagetumors are potentially curable with surgery, chemotherapy, orradiotherapy, and late stage patients usually receive chemotherapy orbest supportive care. Intermediate stage or locally advanced NSCLC,which comprises 25% to 30% of all cases of NSCLC, is more typicallytreated with multimodality therapy. This is a stage of tumor developmentwhen angiogenesis is a very important factor. New blood vessels areneeded to support further tumor growth and for the development ofmetastases. Therefore, this stage is amenable to treatment withantiangiogenic agents to prevent the development of new blood vessels.The efficacy of this therapy can be further improved by the combinationof the antiangiogenic therapy with cytotoxic chemotherapy or radiationtherapy to eliminate existing tumor.

Breast cancer also presents treatment difficulties using known agents.The incidence of breast cancer in the United States has been rising at arate of about 2%/year since 1980, and the American Cancer Societyestimated that 182,000 cases of invasive breast cancer were diagnosed in1995. Breast cancer is usually treated with surgery, radiotherapy,chemotherapy, hormone therapy, or combinations of the various methods.Like other solid tumors, breast cancer requires the development of newblood vessels to support its growth beyond a certain size, and at thatstage in its development, it will be amenable to treatment withantiangiogenic agents.

A major reason for the failure of cancer chemotherapy in breast canceris the development of resistance to the cytotoxic drugs. Combinationtherapy using drugs with different mechanisms of action is an acceptedmethod of treatment which prevents development of resistance by thetreated tumor. Antiangiogenic agents are particularly useful incombination therapy because they are not likely to cause resistancedevelopment since they do not act on the tumor, but on normal hosttissue.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a method for treatingmalignant and cancerous tumors using squalamine, in combination withother, conventional cancer treating agents. In one aspect of theinvention, CNS tumors are treated; in another aspect, lung tumors aretreated; and in yet another, breast tumors are treated.

In one method according to the invention, squalamine is used incombination with conventional cancer treatments to treat tumors. Thetumor is treated by administering an effective amount of a cytotoxicchemical compound in a first treatment procedure, and an effectiveamount of squalamine is administered in a second treatment procedure.

In this method, the cytotoxic chemical compound used in the firsttreatment procedure is a conventional cancer treating agent. Preferableagents include a nitrosourea, cyclophosphamide, adriamycin,5-fluorouracil, paclitaxel and its derivatives, and cisplatin andrelated platinum compounds. These conventional cancer treating agentsare well known to those skilled in this art. Note, M. C. Wiemann andPaul Calabresi, "Pharmacology of Antineoplastic Agents," MedicalOncology, Chapter 10, edited by Paul Calabresi, et. al., McMillanPublishing (1985). Medical Oncology is entirely incorporated herein byreference. One particularly preferred nitrosourea is BCNU, which also isknown as carmustine. Another preferred cytotoxic agent is cisplatin, andyet another is cyclophosphamide. Other conventional cytotoxic chemicalcompounds, such as those disclosed in Medical Oncology, supra., can beused without departing from the invention.

The cytotoxic chemical compound administered in the first treatment stepmay be administered by any conventional technique used in the art (e.g.,oral, subcutaneously, intralymphatically, intraperitoneally,intravenously, or intramuscularly). In one embodiment of the invention,the cytotoxic chemical compound (preferably BCNU, cisplatin, orcyclophosphamide) is administered intravenously. Likewise, squalaminecan be administered by any conventional administration method known inthe art, such as those mentioned above. Subcutaneous injections ofsqualamine one or two times a day are used in one embodiment of thisinvention. Intravenous administration of squalamine one or two times aday are used in another embodiment of the present invention.

The first treatment procedure with the cytotoxic chemical compound maytake place prior to the second treatment procedure (using squalamine),after the second treatment procedure, or at the same time as the secondtreatment procedure. Furthermore, the first treatment procedure may becompleted before the second treatment procedure is initiated (or viceversa). In one embodiment of the invention, the first treatmentprocedure is a one time intravenous administration of a cytotoxicchemical compound (e.g., BCNU, cisplatin, or cyclophosphamide), and thesecond treatment procedure involves daily subcutaneous injections ofsqualamine.

In a second method for treating a tumor according to the invention, thefirst treatment procedure is a radiation treatment, which may be one ormore conventional radiation modalities, using a conventional radiationtreatment regimen known to those skilled in the art. The tumor isexposed to radiation in this first treatment procedure. In a secondtreatment procedure, an effective amount of squalamine is administeredto treat the tumor. Appropriate timing of the radiation treatmentprocedure with respect to the squalamine treatment regimen can bedetermined by those skilled in the art through routine experimentationin order to provide effective tumor treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other advantageous features of the invention will be morefully appreciated when considered based on the following detaileddescription and the attached drawings, wherein:

FIG. 1 shows the general structural formula of squalamine;

FIG. 2 shows a general overview of the angiogenesis process;

FIG. 3 is a drawing used to illustrate the sodium hydrogen exchanger(NHE) process;

FIG. 4 illustrates the effects of conventional amilorides on inhibitingvarious isoforms of mammalian NHEs;

FIGS. 5a and 5b illustrate the effect of squalamine on NHE isoform 3(NHE3) and NHE1 inhibition, respectively;

FIGS. 6a to 6c show the results of a pharmacokinetic study relating tosqualamine;

FIG. 7 illustrates squalamine distribution in various tissues after i.v.administration;

FIG. 8 shows an angiogenesis index using squalamine as determined in therabbit corneal micropocket assay;

FIG. 9 shows the inhibitory effect of squalamine on growth ofendothelial cells as compared to tumor cell lines;

FIG. 10 illustrates survival test results using squalamine in a gliomalethality study with a rat 9L glioma introduced into the brains ofhealthy rats;

FIG. 11 shows the survival of mice carrying human MX-1 breast tumorxenografts and treated with squalamine subsequent to cyclophosphamidetreatment;

FIG. 12 depicts the inhibition of a human lung adenocarcinoma (H460) ina mouse xenograft-combination therapy study with squalamine andcisplatin; and

FIG. 13 illustrates the number of lung metastases following variouschemotherapeutic treatment procedures in mice with subcutaneousimplanted Lewis lung carcinomas.

DETAILED DESCRIPTION OF THE INVENTION

Squalamine has been recognized to have angiogenesis inhibiting activity,i.e., it inhibits the formation of blood vessels. Therefore, it isbelieved that squalamine, as an antiangiogenic agent, will be effectivein treating certain diseases or ailments which depend onneovascularization. For example, squalamine may be used for treatingsuch disparate conditions as solid tumor cancers, macular degeneration,diabetic retinopathy, psoriasis, or rheumatoid arthritis, all of whichrequire a separate and new blood flow.

In addition, squalamine can selectively inhibit certain sodium/protonexchangers (also called "NHEs" or "proton pumps" in this application).Several different isoforms of NHE are known to exist in mammals (e.g.,NHE1, NHE2, NHE3, NHE4, and NHE5). Squalamine has been found tospecifically inhibit NHE3 and not NHE1 or NHE2. Accordingly, squalaminemay be used for treating proliferation or activation dependentconditions which rely on the function of NHE3, such as cancer, viraldiseases, and ischemic reprofusion injury.

Further studies with squalamine and NHE have demonstrated thatsqualamine acts on a very specific portion of the NHE3, namely the 76carboxyl-terminal amino acids of the molecule. If this portion of theNHE3 molecule is removed, squalamine has virtually no effect on theactivity of the molecule, even though the molecule is still active as asodium/proton exchanger.

Applicants have discovered still further uses of squalamine.Specifically, applicants have found that squalamine in combination withconventional cancer treating agents, e.g., cytotoxic chemical compoundsand radiation treatments, will decrease the size and growth of tumors.Even more significantly, applicants have found that the combinationdecreases the growth rate of highly proliferative CNS tumors, lungtumors, and breast tumors and can confer survival advantages.

In the practice of this aspect of the invention, a cytotoxic chemicalcompound is used in a first tumor treatment procedure, and squalamine isused in a second tumor treatment procedure. The first and secondtreatments may be administered in any time sequence or evensimultaneously. In another embodiment, two or more cytotoxic chemicalagents may be administered simultaneously or sequentially in the firsttreatment process.

The cytotoxic chemical compound(s) used in the first treatment proceduremay be any conventional agent, but it is preferably one of the followingagents: a nitrosourea, cyclophosphamide, adriamycin, 5-fluorouracil,paclitaxel and its derivatives, and cisplatin and related platinumcompounds. These materials are conventional cancer treating agents whichare known to those skilled in this art, as set forth in MedicalOncology, supra. One particularly preferred nitrosourea is BCNU, whichis also known as "carmustine" or "1,3-Bis(2-chloroethyl)-1-nitrosourea."Cyclophosphamide also is known asN,N-Bis-(2-chloroethyl)-N'-(3-hydroxypropyl)phosphordiamidic acid cyclicester monohydrate. Adriamycin also is known as doxorubicin.

Paclitaxel is available under the tradename "Taxol." Various derivativesof paclitaxel may be used in accordance with the invention, such astaxotere or other related taxanes. Cisplatin, another of the cytotoxicchemical compounds which may be used in accordance with the invention,also is known as cis-Diamminedichloroplatinum. Those of ordinary skillin the art would be familiar with other specific cytotoxic agents thatcould be used in the process of the invention.

There are no limitations on the chemotherapeutic agent that can be usedin this invention. Other conventional chemotherapeutic agents that canbe used with squalamine in the process of the invention includemethotrexate, thiotepa, mitoxantrone, vincristine, vinblastine,etoposide, ifosfamide, bleomycin, procarbazine, chlorambucil,fludarabine, mitomycin C, vinorelbine, and gemcitabine.

The first and/or second treatments may be administered by any suitabletechnique, such as oral, "s.q.," "i.p.," "i.m.," "i.l.," or i.v." Inthis application, the terms "s.q.," "i.p.," "i.m.," "i.l.," and "i.v."will be used to refer to subcutaneous administration of squalamine orother substances, intraperitoneal administration of squalamine or othersubstances, intramuscular administration of squalamine or othersubstances, intralymphatic administration of squalamine or othersubstances, and intravenous administration of squalamine or othersubstances, respectively.

In one embodiment, BCNU is delivered to a patient first as a one timeintravenous dosage, and thereafter squalamine is injected s.q. twicedaily. In another embodiment, cyclophosphamide is the cytotoxic agent.In another embodiment, cisplatin is the cytotoxic agent. If appropriate,the cytotoxic chemical compound and the squalamine may be deliveredsimultaneously by a common pharmaceutical carrier (e.g., one injectionincluding both squalamine and the cytotoxic chemical compound). Otherappropriate combinations of administration techniques may be usedwithout departing from the invention. Those skilled in the art will beable to ascertain the appropriate treatment regimens, depending on thecytotoxic chemicals used, the dosages, etc., through routineexperimentation.

The squalamine treatment procedure in accordance with the invention alsomay be used with radiation treatment (e.g., cobalt or X-ray treatment)as the first treatment procedure. In this embodiment of the invention,the first treatment procedure is a radiation treatment, and the secondtreatment procedure is squalamine administration. Radiation treatmentscan proceed on a schedule in combination with the squalamine treatmentsto provide optimum results. Such scheduling of the treatment procedurescan be ascertained by the skilled artisan through routineexperimentation. Any conventional radiation treatment, such as thosedescribed in Medical Oncology, supra., may be used without departingfrom the invention. In addition to radiation and squalamine treatments,the tumor also may be treated with one or more cytotoxic chemicalcompounds in a third treatment procedure.

The invention will be described below in terms of various specificexamples and preferred embodiments. These examples and embodimentsshould be considered to be illustrative of the invention, and not aslimiting the same.

I. PHYSIOLOGICAL PROPERTIES OF SQUALAMINE

A. Antiangiogenic Activity

Squalamine has been demonstrated to be useful as an antiangiogenicagent, i.e., squalamine inhibits angiogenesis. Angiogenesis, the processof forming new blood vessels, occurs in many basic physiologicalprocesses, such as embryogenesis, ovulation, and wound healing.Angiogenesis also is essential for the progression of many pathologicalprocesses, such as diabetic retinopathy, inflammation, and malignancy(tumor development). In view of its antiangiogenic properties,squalamine may be used for treating various ailments and conditionswhich depend on angiogenesis, such as those identified above.

Angiogenesis is a multiple step process which is schematicallyillustrated in FIG. 2. First, endothelial cells must become activated,for example, by attaching a growth factor such as vascular endothelialgrowth factor ("VEGF") or basic-fibroblast growth factor ("b-FGF"). Thecells then move, divide, and digest their way into adjacent tissuethrough the extracellular matrix. The cells then come together to formcapillaries and lay down new basement membrane. This angiogenesisprocess is illustrated in the upper portion of FIG. 2. Each of thesedevelopment stages during angiogenesis is important and may be affectedby antiangiogenic agents.

Certain compounds which are believed to be antiangiogenic compounds(e.g., matrix metalloproteinase inhibitors, such as minocycline, SU101or marimistat) act at later stages in this multistep angiogenesisprocess. These compounds will be referred to as "downstream"angiogenesis inhibitors. For a discussion of matrix metalloproteinaseinhibitors, please refer to Teicher, Critical Reviews inOncology/Hematology, Vol. 20 (1995), pp. 9-39. This document is entirelyincorporated herein by reference. In contrast to these knownantiangiogenic compounds, squalamine acts at a very early stage in theprocess by inhibiting the cell activation action of growth factors,i.e., it is an "upstream" angiogenesis inhibitor. As shown in FIG. 2(toward the bottom), squalamine inhibits the sodium-proton pumps thatare normally active and activated by the growth factors. Inhibition ofthe proton pump places the cell in a quiescent state, and, in this way,capillary formation and angiogenesis is impeded. In effect, the growthfactor signal is aborted in the presence of squalamine.

B. Capillary Regression Activity

In addition to antiangiogenic characteristics, squalamine has been shownto have a capillary regression effect in newly formed capillaries. A onetime dose (100 ng) of squalamine was applied to capillary beds of youngchick embryos that were 2-3 days old. After five minutes, this dose ofsqualamine appeared to have little effect on the capillary beds. Intwenty minutes, however, the capillary bed appeared to be disappearing(i.e., the vessels appeared to be closed off). After forty minutes,additional capillary regression was observed.

The capillary bed also was observed after sixty minutes. At this time,it was noted that some of the capillary vessels were beginning tore-appear, but only the more major vessels were re-appearing. The smallvessels were not re-appearing at that time. Four to five days after theone time squalamine treatment, the effect of the squalamine dose was nolonger apparent, but newly formed capillaries in the embryos remainedsusceptible to squalamine induced regression for a limited time whilethey were newly formed.

From this test, applicants concluded that squalamine-induced capillaryregression is reversible, at least with respect to certain capillaries.It also was concluded that squalamine is more effective against smallmicrocapillary blood vessels (i.e., the microvascular bed) as comparedto the major blood vessels. Close histological examination of chickmicrovessels exposed to squalamine revealed vessel occlusion was due toshrinkage of endothelial cell volumes in cells wrapped around the vessellumen. The applicants postulate that occlusion or regression of smallblood vessels by squalamine significantly contributes to the ability ofsqualamine to impede the flow of nutrients and growth factors intotumors and thereby slows or blocks the rate of growth of the tumors.

C. NHE Inhibitory Activity Of Squalamine

Cell growth and division is necessary for blood vessel and capillarygrowth and formation. Capillary formation requires a specificextracellular matrix. The NHE antiporter system of a cell is connectedto the extracellular matrix. Activation of the NHE antiporter isnecessary to induce cell growth, and interference with the NHEantiporter interrupts the matrix signal and interferes with cell growth.When endothelial cell growth is interrupted, capillary growth isimpeded.

The NHE antiporter of cells may be activated in different ways. Forexample, insoluble fibronectin activates the NHE antiporter byclustering and immobilizing Integrin α_(v) β₁, independent of the cellshape (the growth of anchorage-dependent cells requires both solublemitogens and insoluble matrix molecules). In addition, the attachment ofstimuli to the extracellular matrix or cell attachment events involvingviruses also activate the NHE antiporter.

When activated, the NHE antiporter induces cell growth by regulating thepH of the cell. As shown in FIG. 3, the chloride-bicarbonate exchangerand NHE are complementary pH regulators in cells. Thechloride-bicarbonate exchanger makes the cell become more alkaline,while NHE contributes to the control of hydrogen ion concentration inthe cell. When the NHE is inhibited, the cells become acidic (lower pH)and growth stops. This does not mean that the cell dies; it means onlythat the cell enters a quiescent state (i.e., it does not divide). Ifthe cell returns to a normal pH, growth may resume. When the NHE isactivated, the cell becomes more alkaline (higher pH), it pumps outprotons, and growth proceeds. Interaction of various modulatory factors(e.g., serum components, secondary messengers, etc.) with one portion ofthe cytoplasmic region of NHE activates the antiporter, whileinteraction with another portion inhibits the antiporter. These portionsof NHE are described in Tse, et al., "The Mammalian Na⁺ /H⁺ ExchangerGene Family--Initial Structure/Function Studies," J. Am. Soc. Nephr.,Vol. 4 (1993), pg. 969, et seq. This article is entirely incorporatedherein by reference.

Sodium-proton pumps (NHEs) are responsive to different growth stimuliwhich activate the pump. As noted above in connection with FIG. 2, theproton pump may be activated by attachment of growth factors (e.g., VEGFand b-FGF) to the cell. Additionally, as shown in FIG. 3, other stimuli,such as virus attachment, addition of various mitogens, sperm attachmentto an egg, etc, also can cause NHE activation and alkalinization of thecell. Attachment of these stimuli to the extracellular matrix activatesthe NHE antiporter of the cell and induces cell growth.

At least five different mammalian isoforms of NHE exist, and each has adistinct tissue distribution. Nonetheless, all act in the same manner.NHE1 is the antiporter found in all tissues. NHE2 and NHE3 are morerestrictive in their tissue distribution.

The effect of squalamine on NHE activity was measured to determine whichisoforms of NHE were affected by squalamine. NHE activity can bemeasured under various different cellular conditions. Acid loading acell activates all of the antiporters and permits measurement of NHE.NHE activity also can be measured after growth factor stimulation of thecell. Additionally, the NHE activity can be measured when the cell is inan unstimulated state, because the antiporters, even if unstimulated,continue to function at a slow, but non-zero rate. In each of thesecellular conditions, NHE activity usually is measured in the absence ofbicarbonate.

Amilorides, which are the classic inhibitors of activated NHEantiporters and which act as direct competitive inhibitors of Na⁺ ionbinding to NHE, do not turn off the antiporter activity in unstimulatedcells. As illustrated in FIG. 4, amiloride and amiloride analoguesspecifically act against NHE1 over NHE2 or NHE3. NHE3 in particular isrelatively resistant to inhibition by the amilorides. In contrast to theamilorides, when NHE1 activity was measured in unstimulated melanomacells, applicants found that squalamine substantially down regulates theactivity of the antiporter.

The following describes the test used to determine that squalamineinhibits NHE3, but not NHE1 or NHE2. NHE deficient fibroblast cells(PS120) transfected with an individual human NHE gene were loaded with apH sensitive dye 2'7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein(BCECF). NHE activity was measured by spectrofluorometric methods usingthis dye and by amiloride sensitive isotopic ²² Na⁺ cellular uptake. Thecells were acidified by exposure to ammonium chloride in the absence ofsodium to eliminate sodium and deactivate the proton pumps. The ammoniumchloride was washed out by exposing the cells to tetramethyl ammoniumchloride in bicarbonate free medium. The cells were consequentlyacidified, but in the absence of sodium, the NHE ion pumps did notactivate. For this test, as shown in FIGS. 5a and 5b, 7 μg/ml ofsqualamine was added to the cells in each case. Sodium then was addedback at various concentrations (see the abscissa of FIGS. 5a and 5b) todrive the antiporters (human NHE3 in FIG. 5a and human NHE1 in FIG. 5b).The antiporters were driven at different rates, as evidenced by thecellular pH change rate, depending on the amount of sodium added. Asshown in FIG. 5a, when measuring the effect of squalamine on the humanNHE3 antiporter, the pH change rate was lower in the squalamine treatedcells than the pH change rate in the control group (without squalamine).This indicates that squalamine inhibits human NHE3. In FIG. 5b, however,there is no effective difference in the pH change rate between thesqualamine treated samples and the control when measuring the human NHE1antiporter. From these tests, applicants concluded that squalamineinhibits human NHE3, but not human NHE1. Additionally, in similar tests,it was found that rabbit NHE1 and NHE2 are not affected by squalamine,but rabbit NHE3 is inhibited by squalamine treatment.

In the transfected cells used in this test, it took at least 30 minutesbefore the NHE3 inhibition effect induced by squalamine was observed.Thus, squalamine did not act like the classic NHE inhibitor amiloride oranalogues of amiloride, which are direct competitive inhibitors forsodium and, therefore, act rapidly as NHE inhibitors. Furthermore, itwas observed that the NHE inhibiting effect of squalamine occurred inthe absence of lactase dehydrogenase (LDH) leakage from the cell.Because LDH leakage is a non-specific marker of cytotoxicity, it wasconcluded that squalamine does not have a general cytotoxic effect.

This NHE3 inhibiting activity of squalamine has been mapped to the 76C-terminal amino acids on the NHE3 molecule. If the 76 C-terminal aminoacids of rabbit NHE3 are removed from the molecule, squalamine has beenfound to have virtually no effect on the activity of the molecule, whilethe molecule remains active as a sodium/hydrogen exchanger. Thus, the 76C-terminal amino acids of NHE3 are the site of inhibition by squalamine.It is believed that the squalamine effect on these accessory proteins ofNHE3 is tied to an inhibitory effect on tyrosine kinase-dependentactivity, although applicants do not wish to be bound by any specifictheory of operation.

As noted above, it has been concluded that squalamine inhibits NHE3 andnot NHE1. This inhibitory effect of squalamine, however, has been foundto work in a manner different from classical and known NHE3 inhibitors.In contrast to squalamine, other inhibitors of NHE3 (e.g., amiloride,amiloride analogues, genestein, calmodulin, and protein kinase C) alsoinhibit NHE1. Such inhibitors affect only the absolute number of protonsthat can be secreted by the cell (i.e., "V_(max) "), if one looks at thekinetic characteristics of the inhibition. Squalamine, on the otherhand, not only inhibits V_(max), but it also forces the cell to fall toa lower pH, as evidenced by a reduction in the Km value. Note thefollowing Table 1, which correlates to data collected in the test ofFIG. 5a.

                  TABLE 1                                                         ______________________________________                                                  Squalamine (7 μg/ml)                                                                   Control                                                 ______________________________________                                        Km          0.338         0.595                                               n           1.88          1.22                                                V.sub.max   1282          2958                                                ______________________________________                                    

Thus, squalamine inhibits NHE with nonallosteric kinetics (i.e.,nonclassical allosteric inhibition). In additional tests, it also wasfound that squalamine (at a 1 hour pretreatment) decreased the V_(max)of rabbit NHE3 in a concentration dependent manner (13%, 47%, and 57%with 1, 5, and 7 μg squalamine/ml, respectively). This observedsqualamine effect on the V_(max) was time dependent, with a maximumeffect occurring at one hour exposure. The observed effect was fullyreversible within three hours after removing the cells from the medium.

In view of the test results relating to the effect of squalamine onNHE3, applicants believe that NHE3 is important in maintaininghomeostasis of the unstimulated cell. The applicants further believethat prevention of cellular activation by squalamine, especiallyactivation of endothelial cells or precursor cells which participate information of new blood vessels during pathophysiological vascularization(such as during tumor growth), is the mechanism through which squalamineinhibits tumor growth.

Applicants have further observed that squalamine changes endothelialcell shape. This suggests that transport proteins which control cellvolume and shape may be a squalamine target.

Additional testing of squalamine has indicated that squalamine inhibitedbrush border membrane vesicle (BBMV) NHE only when the tissue waspretreated with squalamine (51% inhibition at 30 minutes exposure).Direct addition of squalamine to PS120 fibroblasts during measurement ofthe exchanger activity had no effect.

D. Pharmacokinetic Studies Of Squalamine

A pharmacokinetic study of squalamine was performed to ascertain theresidence time of squalamine in the body. FIGS. 6a to 6c illustrate thetest results where squalamine was administered subcutaneously (50 mg/kg,FIG. 6a), intraperitoneally (dose 240 μg; 10 mg/kg, FIG. 6b), andintravenously (10 mg/kg, FIG. 6c). The half-life of squalamine whengiven intravenously (FIG. 6c) was acceptable (35 minutes), but it waseven higher when it was administered intraperitoneally (FIG. 6b,half-life=172 minutes) and subcutaneously (FIG. 6a, half-life=5.6hours).

In addition to these squalamine half-life tests, applicants have testedto ascertain the distribution of squalamine in a mouse after intravenousadministration. FIG. 7 illustrates the distribution of squalamine inmouse tissue two hours after i.v. administration. Some squalamine iscontained in most of the tissues, with most of the squalamineconcentrating in the liver and the small intestine. The test resultsshown in FIG. 7 indicate good squalamine distribution. Notably, however,not much squalamine is present in brain tissue. From this, applicantsconclude that squalamine probably does not cross the brain/bloodbarrier. In treating brain tumors, it is believed that the squalamineacts on the endothelial cells in the brain, and in this way, it need notcross the brain/blood barrier

The following examples describe more detailed experiments used to testthe antiangiogenic characteristics of squalamine in the process of theinvention.

EXAMPLE 1 Rabbit Corneal Micropocket Assay

In determining whether a compound is antiangiogenic, the rabbit cornealmicropocket assay is an accepted standard test. In this test, anincision is made in one rabbit cornea, and a stimulus is placed in theincision. The stimulus is used to induce blood vessel formation in thenormally avascular corneal region. As one example, a solid tumor in apolymeric matrix can be placed in the cornea as the stimulus because thetumor will release a number of angiogenic growth factors to stimulatenew capillary growth. The tumor-derived angiogenic growth factorsstimulate the endothelial cells at the scleral junction in the eye toinitiate blood vessel growth toward the stimulus. A second polymerpellet (e.g., an ethylene/vinyl acetate copolymer) is placed between thescleral junction and the stimulus. This polymer pellet is either empty(a negative control test pellet), or it contains a compound whoseantiangiogenic characteristics are to be tested. The polymer pellet isused to provide a controlled release of the material to be studied.Because of the avascular cornea background in the rabbit cornea, one canvisually assess the results qualitatively. In addition, the number ofblood vessels can be counted and their length, etc., can be measured toprovide a more quantitative evaluation of the results.

The VX2 rabbit carcinoma was implanted in 26 rabbit eyes, in thenormally avascular corneal region, to act as an angiogenesis stimulus.Squalamine was incorporated into a controlled release ethylene/vinylacetate copolymer (20% squalamine and 80% polymer by weight). The loadedpolymer pellets were placed in 13 of the corneas to provide a sustainedlocal release of squalamine. Polymer blanks were provided in theremaining 13 eyes as a control. In this manner, one eye of each rabbitserved as the squalamine test eye and the other eye of the same rabbitserved as the control eye. The eyes were examined weekly using a slitlamp stereomicroscope for three weeks after tumor implantment, and theAngiogenesis Index ("AI") was calculated (this calculation will bedescribed in more detail below with reference to FIG. 8). The squalamineloaded polymer was found in vitro to release active squalaminethroughout the treatment period. After the test, the corneas wereexamined histologically.

Using this test, squalamine was found to be a potent inhibitor of tumorinduced capillary formation. Fewer blood vessels were observed in thecornea treated with squalamine as compared to the control cornea, andthese vessels were generally shorter than the vessels in the controlcornea.

Some of the corneas were then sectioned to observe the effect ofsqualamine on the tumor cells themselves. The untreated control corneashad many vessels in and adjacent to the tumor. The tumors in thesqualamine-treated corneas were still viable (i.e., the tumors were notdead), but there was essentially no vasculature associated with thosetumors. Thus, the squalamine-treated tumors had greatly diminishedvascularity as compared to the corresponding control tumor sections.These findings suggested that squalamine works against the bloodvessels, and not against the tumor itself.

FIG. 8 shows a graphical representation of the results of the rabbitcornea micropocket assay test. To provide a quantitative evaluation, theAngiogenesis Index ("AI") of each eye was determined. To determine theAngiogenesis Index, first the vessel density ("D_(vessel) ") in an eyewas graded on a 0-3 scale as follows:

                  TABLE 2                                                         ______________________________________                                        D.sub.vessel Value Determinations                                             D.sub.vessel Value                                                                       Visual Observation                                                 ______________________________________                                        0          No vessels present                                                 1          1-10 vessels present                                               2          >10 vessels present, but loosely                                              packed                                                             3          >10 vessels present, packed densely                                ______________________________________                                    

The vessel length ("L_(vessel) ") was then measured in each cornea. Thevessel length is the length of the longest vessel measured from thecornea-scleral junction to the distal edge of the longest vessel growth.The Angiogenesis Index then is determined from these measurements by thefollowing equation:

    AI=D.sub.vessel ×L.sub.vessel.

FIG. 8 shows the mean Angiogenesis Index for each group of corneas(squalamine treated and untreated) in the rabbit cornea micropocketassay after 1, 2, and 3 weeks. As shown in the figure, squalamine wasvery inhibitory to the growth of new blood vessels. The squalaminetreated eyes showed a significantly reduced AI value as compared to theuntreated eyes (37% reduced at Day 14 (p=0.05, Wilcoxon rank sum test)and 43% reduced at Day 21 (p<0.01). This data illustrates thatsqualamine inhibits tumor induced growth of new blood vessels orcapillaries over a long time period. More specifically, squalamineexhibits high antiangiogenic activity even after three weeks.

EXAMPLE 2 Squalamine Does Not Cause Inflammation

In the rabbit corneal micropocket assay test, if the rabbit corneabecomes inflamed, this inflammation can lead to the formation of newblood vessels in the cornea. Such inflammation would skew the testresults. Therefore, tests were conducted to determine whethersqualamine, in and of itself, was responsible for any inflammatoryresponse in the cornea. Several non-bioresorbable ethylene/vinyl acetatecopolymer pellets were loaded with different concentrations ofsqualamine, namely, 2%, 10%, and 20% squalamine, by weight. Thesepellets were then placed in rabbit corneas which did not include anangiogenic stimulus. Squalamine did not induce inflammation at any ofthese concentrations. Thus, squalamine does not lead to the generationof new blood vessels by inflaming the cornea.

EXAMPLE 3 Squalamine Use in Brain Tumor Treatment

The rabbit corneal micropocket assay test results suggested toapplicants that squalamine may be a potent antiangiogenic agent thatinhibits neovascularization. Recognizing that the exponential growth ofsolid tumors in the brain is dependent on neovascularization, applicantsassessed the activity of squalamine in an animal model on the growth ofsolid tumors in the brain.

Of solid brain tumors, malignant gliomas are the most common form ofcancerous tumors. These tumors are the third leading cause of death fromcancer in young adults between the ages of 15 and 34. Malignant gliomasare characterized by their ability to induce the normally quiescentbrain and/or CNS endothelial cells into a highly proliferative andinvasive state. The gliomas express vascular endothelial growth factor("VEGF") and other growth factors which stimulate inducible receptors onCNS endothelial cells in a paracrine manner (i.e., the VEGF originatesfrom the tumor cell and stimulates the endothelial cells). The CNSendothelial cells subsequently initiate angiogenic invasion and thusprovide nourishment of the glioma. Applicants tested the antiangiogenicactivity of squalamine against gliomas by testing (1) its ability toselectively inhibit VEGF-mediated stimulation of endothelial cells and(2) its effect against experimental murine glial tumors.

In vitro tests were first performed to determine that squalamine actsspecifically on endothelial cells. Applicants used endothelial cellsbecause such cells are involved in the early steps of angiogenesis, asdescribed above in conjunction with FIG. 2. Specifically, tumorangiogenesis is a series of sequential and overlapping steps. First, theendothelial cells activate and proliferate. Then, proteolytic enzymesare produced and the cells migrate. New basement membranes must then begenerated. In this manner, new blood vessels are generated and tumorsize increases.

In conducting this in vitro analysis, the following cell lines weretested: (a) bovine retinal endothelial cells; (b) 9L and C6 rat gliomacells; (c) human H80 glioma cells; and (d) VX2 rabbit carcinoma cells(the same type as the tumors implanted in the rabbit corneal micropocketassay test described above). The endothelial mitogen which was used inthis analysis was VEGF at a concentration of 20 ng/ml.

The cells were allowed to attach overnight to tissue culture platescontaining an optimized growth media. Following attachment, the cellswere exposed to solvent only or to increasing concentrations ofsqualamine (0, 10, 20, 30, 60, and 90 μg squalamine/ml). Cell growth wascounted daily for three days using a Coulter Counter. A total of 10,000cells per well were plated and each experimental concentration wastested in quadruplicate. The results were then averaged. The bovineretinal endothelial cells were grown and treated in an identical mannerto the other cell lines, except that the growth of these cells wasmeasured after the addition of 20 ng/ml of human recombinant VEGF to thecells prior to the squalamine treatment.

Cell proliferation by all tumor lines and by endothelial cells nottreated with VEGF was statistically unaffected after exposure for 24 and48 hours to squalamine concentrations up to 30 μg/ml. Growth of theVEGF-stimulated endothelial cells, however, was significantly reduced bysqualamine at these same times in a concentration dependent manner.Percentage endothelial cell growth inhibition (%I) was determined by thefollowing equation: ##EQU1## The following Table shows the results at 48hours for the VEGF-stimulated endothelial cell line.

                  TABLE 3                                                         ______________________________________                                        Percent Inhibition Data                                                       Squalamine Conc.                                                                            % Inhibition (average)                                          ______________________________________                                        10 μg/ml   38% (p < 0.01)                                                  20 μg/ml   57% (p < 0.001)                                                 30 μg/ml   83% (p < 0.001)                                                 ______________________________________                                    

Additional data is illustrated in FIG. 9. This figure shows the growthof the various cell lines as a percentage of the growth in the controlgroups for in vitro administration of squalamine at 30 μg/ml after 1, 2,and 3 days. As shown in FIG. 9, growth is reduced for theVEGF-stimulated endothelial cells specifically, while the growth in theother cell lines (H80, C6, and VX2) is not dramatically affected.

Based on this information, applicants concluded that squalaminedramatically and specifically inhibits VEGF-stimulated growth ofendothelial cells in vitro. Thus, squalamine is a potent inhibitor oftumor-induced angiogenesis, and this effect appears to be precipitatedthrough specific inhibition of endothelial cell proliferation induced byVEGF. Thus, squalamine is believed to be well suited for reducing ordiminishing the neovasculature induced by tumors for use in tumorspecific antiangiogenic therapy.

In addition to inhibiting VEGF-stimulated growth of endothelial cells,squalamine also has been found to interfere with growth stimulation inhuman brain capillary endothelial cells induced by b-FGF, PDGF_(bb),scatter factor (HGF or hepatocyte growth factor), conditioned tumormedia, and human brain cyst fluid. Thus, as the tumor puts out a varietyof different growth factors, squalamine has an inhibitory effect onseveral.

In view of these test results, applicants tested squalamine in an animalmodel for brain cancer. To test the effect of squalamine on tumorslocated in the brain, small sections (1 mm³) of existing rat gliomaswere taken from rat flanks where they were being maintained and wereimplanted into the rat brains in two groups of rats. Thus, in thismodel, the tumors were viable when placed in the rat brain. Three daysafter implantation, and after some vasculature had developed, treatmentwith 20 mg/kg/day of squalamine (i.p.) was initiated in one group ofrats. The control animals ("vehicle control" in FIG. 10) were given thecarrier vehicle only (no squalamine), and the other animals were treatedwith squalamine ("Squalamine" in FIG. 10). As shown in the figure, theanimals treated with squalamine had a 38% increase in mean survival time(x=24.9 days v. x=18.0 days). FIG. 10 further illustrates that in thisanimal model, the squalamine treated rats, in general, had an increasedsurvival time.

A squalamine toxicity test was performed in another animal model.Conventional cytotoxic chemical compounds are quite toxic. For example,BCNU, which is a conventional chemotherapy agent, has a cumulativetoxicity effect. For this reason, it is administered only one time to apatient. The use of BCNU is described on pages 304 and 305 of Calabresiin Medical Oncology, supra. In order to test the toxicity of squalamine,a group of rats was given a daily squalamine dose of 20 mg/kg/day (i.p.)for more than 30 days and maintained for up to 200 days followingdosing. The animals in this study remained healthy. This resultindicates that squalamine has little or no toxicity.

EXAMPLE 4 Squalamine Use With Conventional Cancer Treatments

As described above, squalamine is an upstream inhibitor of theangiogenesis process by inhibiting the activation of endothelial cellsafter growth factor interaction. Because of its angiogenesis inhibitingproperties, squalamine has been demonstrated to be effective in treatingsolid tumors which rely on neovascularization to proliferate. Applicantstested to determine whether beneficial results could be obtained whentreating tumors by combining a squalamine treatment (an upstreamangiogenesis inhibitor) with a conventional cancer treatment using analkylating agent.

a. The Squalamine 9L Glioma Flank Study

Four groups of rats (twenty total Fisher 344 rats, 200 g) were givens.q. transplants of 1 mm³ 9L gliosarcoma tumors (9L glioma) on Day 0.The tumors were implanted in the rat flanks to avoid complicationsrelating to adequate brain levels of squalamine. Randomization andtreatment began on Day 5 according to the following scheme:

                  TABLE 4                                                         ______________________________________                                        Treatment Conditions                                                          Group No.                                                                            Treatment                                                              ______________________________________                                        1      Saline (control group)                                                 2      One time dose of 14 mg/kg BCNU given i.p. on Day 5                     3      Squalamine - 20 mg/kg given s.q. B.I.D..sup.1                          4      One time dose of 14 mg/kg BCNU given i.p. on Day 5 and                        daily injection of squalamine - 20 mg/kg given s.q. B.I.D-                    beginning on Day 5.                                                    ______________________________________                                         .sup.1 The term "B.I.D" means that the component is administered twice a      day (10 mg/kg given at two different times each day).                    

On Day 25 or 26 after tumor implantation, the tumor size was measureddirectly. The tumor size (i.e., its volume "V") was estimated based onvolumetric calculations determined from the measured length ("L"), width("W"), and height ("H") of the tumor (V_(tumor) spheroid ≈0.5×L×W×H).Table 5 summarizes the results. The tumor volumes shown in Table 5represent the mean tumor volumes for each treatment group for thoseanimals that survived to the end of the experiment.

                  TABLE 5                                                         ______________________________________                                        Tumor Values                                                                                    Mean Tumor volume                                                                           % Reduction (based                            Group No.                                                                            No. of Animals                                                                           (mm.sup.3)    on control volume)                            ______________________________________                                        1      5          18,324        --                                            2      6          2,547         86.1%                                         3      5          3,347         81.7%                                         4      4          38            99.8%                                         ______________________________________                                    

Table 5 illustrates the advantageous results achieved when treatingtumors with the combination of squalamine and the nitrosourea BCNU(Group 4). A 99.8% reduced mean tumor size was observed when treatingwith both squalamine and BCNU in this group. Table 5 further shows thatsqualamine alone (Group 3) was effective in treating the tumor. Thetumor size was reduced by 81.7% in Group 3, as compared to the controlgroup.

Applicants conclude that the use of squalamine in combination withconventional cytotoxic chemical compounds can slow or halt the spread ofbrain cancers. The tumor itself shrinks and becomes necrotic. It isexpected that combined squalamine and cytotoxic chemical treatment willextend survival. Thus, this treatment potentially will allow managementof brain cancers.

b. Squalamine Use in Breast Tumor Treatment

The human MX-1 breast cancer line has previously been used to documentin vivo activity of cyclophosphamide and other cytotoxicchemotherapeutic compounds either as single agents or in combination (T.Kubota, et al., Gann 74, 437-444 (1983); E. Kobayashi, et al., CancerResearch 54,, 2404-2410 (1994); M.-C. Bissery, et al., Seminars inOncology 22 (No. 6, Suppl. 13), 3-16 (1995)). These documents each areentirely incorporated herein by reference. Squalamine was examined asadjunctive therapy following a single 200 mg/kg dose ofcyclophosphamide. The cyclophosphamide was injected on day 14 followingimplantation of the tumor, at a time when the tumors measured 65-125 μl.The cyclophosphamide caused partial regression in all animals andcomplete regression in a small fraction of the animals. The animals werethen randomized to three treatment arms (each n=27): vehicle dosing only(Intralipid); squalamine given 10 mg/kg/day in Intralipid; andsqualamine given 20 mg/kg/day in Intralipid for five days a week.Animals whose tumors exceeded 2 grams at any time during the experimentwere euthanized. The experiment was continued for 90 days afterinitiating squalamine treatment to ensure that only mice experiencinglong-term cures were still alive. The high dose squalamine wasdiscontinued after five weeks of treatment because of animal weight lossand potential toxicity concerns, so these animals did not receivesqualamine for the last eight weeks of the experiment. The low dosesqualamine treatment produced a significant (P<0.01) inhibition in therate of progression of the breast tumors at all times examined (FIG.11). The high dose squalamine treatment produced significant (P<0.05)delay in progression of the breast tumors only at 30 dayspost-initiation (i.e., only while squalamine was still being given), buthigh dose squalamine also doubled the long-term cure rate in theseanimals compared to controls which received cyclophosphamide alone (FIG.11). Examination of the history of the long-term cure animals whichreceived cyclophosphamide and high dose squalamine revealed that theadditive effects of squalamine were manifested within two weeks afterstarting squalamine treatment.

c. Squalamine Use in Lung Tumor Treatment

Studies in a nude mouse xenograft model of lung cancer have been carriedout using several human lung cancer lines which differ in their growthrate. The data collected show that squalamine has synergistic activityin combination with cisplatin (e.g., FIG. 12). The experimental lungcancer model design involves subcutaneous injection of 5×10⁶ tumor cellsfollowed by a single injection of the chemotherapeutic drug on day 3 or4. Daily intraperitoneal squalamine injections with 20% Intralipid as avehicle began the following day for some groups of mice and continueduntil the experiment was terminated 7-14 days later. Groups of micereceiving squalamine alone started receiving the aminosterol on the sameday as aminosterol treatment in the combination chemotherapy groups.Tumor volumes were then determined at termination of the experiment andcompared. It was found for both the aggressively growing H460 human lungadenocarcinoma line and for the more slowly growing Calu-6 human lungadenocarcinoma line that squalamine had minimal effects on tumor growthas a monotherapeutic agent when started on day 4 or 5, but couldcontribute to growth inhibition if it were started on day 1. However,when used starting on day 4 or 5, in combination with cisplatin, givenat or near a maximum tolerated dose, squalamine significantly andreproducibly improved tumor growth inhibition over cisplatin alone in adose-dependent fashion for both the H460 and Calu-6 cell lines.

d. Squalamine Use in Metastatic Lung Cancer

The murine Lewis lung adenocarcinoma was implanted subcutaneously in thehind-leg of male C57BL/6 mice and allowed to grow for one week. Groupsof mice were then left untreated or treated with either squalamine (20mg/kg/day, s.c.), cyclophosphamide (125 mg/kg, i.p. on days 7, 9 and11), cisplatin (10 mg/kg, i.p. on day 7), the combination of squalamineand cyclophosphamide, or the combination of squalamine and cisplatin. Onday 20, the animals were sacrificed, and the mean number of lungmetastases were determined for each group. All treatments reduced thenumber of metastases; however, the most effective treatments were thecombination of squalamine with either of the cytotoxic agents (FIG. 13).

II. THERAPEUTIC ADMINISTRATION AND COMPOSITIONS

The mode of administration of squalamine may be selected to suit theparticular therapeutic use. Modes of administration generally include,but are not limited to, transdermal, intramuscular, intraperitoneal,intravenous, subcutaneous, intranasal, inhalation, intralymphatic,intralesional, and oral routes. The squalamine compounds may beadministered by any convenient route, for example, by infusion or bolusinjection, or by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal, and intestinal mucosa, etc.), and it may beadministered together with other biologically active agents.Administration may be local or systemic.

The present invention also provides pharmaceutical compositions whichinclude squalamine as an active ingredient. Such compositions include atherapeutically effective amount of squalamine and a pharmaceuticallyacceptable carrier or excipient. Examples of such a carrier include, butare not limited to, saline, buffered saline, dextrose, water, oil inwater microemulsions such as Intralipid, glycerol, and ethanol, andcombinations thereof. The formulation of the pharmaceutical compositionshould be selected to suit the mode of administration.

The pharmaceutical composition, if desired, also may contain effectiveamounts of wetting or emulsifying agents, or pH buffering agents. Thepharmaceutical composition may be in any suitable form, such as a liquidsolution, suspension, emulsion, tablet, pill, capsule, sustained releaseformulation, or powder. The composition also may be formulated as asuppository, with traditional binders and carriers, such astriglycerides. Oral formulations may include standard carriers, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc.

Various delivery systems are known and may be used to administer atherapeutic compound of the invention, e.g., encapsulation in liposomes,microparticles, enteric coated systems, microcapsules, and the like.

In one embodiment, the pharmaceutical composition is formulated inaccordance with routine procedures to provide a composition adapted forintravenous administration to humans. Typically, compositions forintravenous administration are solutions in 5% dextrose and sterilewater or Interlipid. Where necessary, the pharmaceutical compositionalso may include a solubilizing agent and a local anesthetic toameliorate pain at the site of an injection. Generally, the ingredientsof the pharmaceutical composition are supplied either separately ormixed together in unit dosage form, for example, as a dry lyophilizedpowder or water-free concentrate in a hermetically sealed container suchas an ampoule or sachette indicating the quantity of active agent. Wherethe pharmaceutical composition is to be administered by infusion, it maybe dispensed with an infusion bottle containing sterile pharmaceuticalgrade water, dextrose, saline, or other pharmaceutically acceptablecarriers. Where the pharmaceutical composition is administered byinjection, an ampoule of sterile water or saline for injection may beprovided so that the ingredients may be mixed prior to administration.

The amount of the therapeutic compound (i.e., active ingredient) whichwill be effective in the treatment of a particular disorder or conditionwill depend on the nature of the disorder or condition, and can bedetermined by standard clinical techniques known to those skilled in theart. The precise dose to be employed in the formulation also will dependon the route of administration and the seriousness of the disease ordisorder, and should be decided according to the judgement of thepractitioner and each patient's circumstances. Effective therapeuticaldoses may be estimated from extrapolations of dose-response curvesderived from in vitro or animal-model test systems.

Suitable dosages for intravenous administration are generally about 1microgram to 40 milligrams of active compound per kilogram body weight.Suitable dosage ranges for intranasal administration are generally about0.01 mg/kg body weight to 20 mg/kg body weight. Suitable dosages fororal administration are generally about 500 micrograms to 800 milligramsper kilogram body weight, and preferably about 1-200 mg/kg body weight.Suppositories generally contain, as the active ingredient, 0.5 to 10% byweight of squalamine. Oral formulations preferably contain 10% to 95%active ingredient.

For use of squalamine as an antiangiogenic or cytotoxic agent or incancer therapies, exemplary dosages are from about 0.01 mg/kg bodyweight to about 100 mg/kg body weight. Preferred dosages are from 0.1 to40 mg/kg body weight.

The invention also may include a pharmaceutical pack or kit includingone or more containers filled with the pharmaceutical compositions inaccordance with the invention. Associated with such containers may be anotice in the form prescribed by a government agency regulating themanufacture, use or sale of pharmaceuticals or biological products,which notice reflects approval by the agency of manufacture, use or salefor human administration.

The conventional cytotoxic chemical compounds used in accordance withthe invention may be present in any suitable form known to those skilledin the art. These chemical compounds also may be administered by anysuitable means also known to those skilled in this art, such as orally,subcutaneously, intravenously, intraperitoneally, intralymphaticly, andintramuscularly.

In describing the invention, applicants have stated certain theories inan effort to disclose how and why the invention works in the manner inwhich it works. These theories are set forth for informational purposesonly. Applicants are not to be bound to any specific chemical orphysical mechanisms or theories of operation.

While the invention has been described in terms of various specificpreferred embodiments and specific examples, those skilled in the artwill recognize that various changes and modifications can be madeithoutdeparting from the spirit and scope of the invention, as defined in theappended claims.

We claim:
 1. A method for treating a tumor that is sensitive to asynergistic combination of a cytotoxic chemical compound and squalamine,comprising the step of: administering a synergistically effective amountof at least one cytotoxic chemical compound in a first treatmentprocedure; and administering a synergistically effective amount ofsqualamine in a second treatment procedure.
 2. A method according toclaim 1, wherein the cytotoxic chemical compound is a member selectedfrom the group consisting of: a nitrosourea, cyclophosphamide,adriamycin, 5-fluorouracil, paclitaxel and its derivatives, cisplatin,methotrexate, thiotepa, mitoxantrone, vincristine, vinblastine,etoposide, ifosfamide, bleomycin, procarbazine, chlorambucil,fludarabine, mitomycin C, vinorelbine, and gemcitabine.
 3. A methodaccording to claim 1, wherein the cytotoxic chemical compound is BCNU.4. A method according to claim 1, wherein the cytotoxic chemicalcompound is cyclophosphamide.
 5. A method according to claim 1, whereinthe cytotoxic chemical compound is cisplatin.
 6. A method according toclaim 1, wherein in the first treatment procedure, the cytotoxicchemical compound is administered intravenously.
 7. A method accordingto claim 6, wherein in the second treatment procedure, the squalamine isadministered subcutaneously.
 8. A method according to claim 6, whereinthe first treatment procedure takes place prior to the second treatmentprocedure.
 9. A method according to claim 1, wherein the first treatmentprocedure is completed before the second treatment procedure begins. 10.A method according to claim 1, wherein the first treatment procedure isa one time injection of the cytotoxic chemical compound.
 11. A methodaccording to claim 10, wherein the cytotoxic chemical compound is BCNU.12. A method according to claim 10, wherein the cytotoxic chemicalcompound is cyclophosphamide.
 13. A method according to claim 10,wherein the cytotoxic chemical compound is cisplatin.
 14. A methodaccording to claim 11, wherein in the second treatment procedure, thesqualamine is administered subcutaneously.
 15. A method according toclaim 1, wherein in the second treatment procedure, the squalamine isadministered orally.
 16. A method according to claim 1, wherein in thesecond treatment procedure, the squalamine is administeredintravenously.
 17. A method according to claim 1, wherein in the secondtreatment procedure, the squalamine is administered subcutaneously. 18.A method according to claim 1, wherein the tumor is a CNS tumor.
 19. Amethod according to claim 1, wherein the tumor is a breast tumor.
 20. Amethod according to claim 1, wherein the tumor is a lung tumor.